I am facing problem in concentrating my protein. I have my protein with 8M urea and it is dilute. I have to concentrate my protein (MW 47kDa) before refolding. I kept my protein sample in dialysis bag (MWCo 3kDa ) in PEG 20,000. It absorbed water and my sample volume got reduced . But when i estimated my sample using bradford reagent my protein concentration was less. I am sure that my protein couldn't come out of that dialysis bag. i doubted whether urea concentration increases and affected my protein during concentrating or for sample with 8M urea will bradford reagent gives good estimation? pls help me ...........
Thank U in advance
8M urea will most likely interfere with this assay. Some references give 3M as a cut-off for not interfering. You can always test it by running a blank with just 8M urea.
The method you used for concentrating could result in a concentration of urea greater than 8M. If you need the protein to remain in 8M urea, I would recommend an Amicon Ultra centrifugal concentrator, sold by Millipore link
. They have very low protein binding and will maintain the concentrated protein in the starting buffer. You can also use these devices for buffer exchange, if you want to.